The light-gated, inwardly rectifying cation channel, channelrhodopsin-2 (ChR2) has become a preferred tool for the targeted light-activation of neurons both in vitro and vivo1-4. Although wild-type (WT) ChR2 can be employed for light-induced depolarization, there is an ongoing search for ChR2 mutants with increased light-sensitivity for potential future clinical applications (WO 03/084994 and 5-7). Higher efficacy would enable depolarization of cell layers distant from the applied light source despite the low optical transmittance of, e.g., brain tissue. An increase in light sensitivity would also solve the problem of potential cell damage under continuous illumination due to the high blue light intensities required for full WT ChR2 activation (1018-1019 ph s−1 cm−2 at 480 nm). Variants with higher light sensitivity are also crucial for research pertaining to the recovery of vision8,9. On the protein level, higher light efficacy can only be achieved by increasing the life-time of the open state and/or by elevating the unit conductance of the channel, as the light sensitivity per se can be improved only marginally due to the nature of the ChR2 chromophore retinal. Previous research has demonstrated that mutations at positions C128 and D156 in helix 3 and 4, respectively, resulted in markedly slowed channel kinetics with open life-times up to 30 minutes and more, yielding a 500-fold or even higher light-sensitivity5,6. These C128 and D156 mutants can be switched off at variable open times by red light. Despite the superior light-sensitivity, their slow closing kinetics remains a limiting factor for their applicability.
Accordingly, there is still a need for light-inducible cation channels exhibiting a higher light sensitivity and faster response kinetics.